Compositions for modulating an xbp1 pathway in a keratinocyte and methods of use

ABSTRACT

Provided herein are compositions and methods for increasing an immune response in a subject, immunizing a subject and/or treating a disease in a subject that relate to keratinocytes. It is a surprising finding of the present invention that modulation of an XBP1 pathway creates a keratinocyte that is itself an immune modulator, or adjuvant. In some embodiments, the concentration of antigen is increased in the vicinity of the keratinocyte, further increasing the immune response by effector cells within that vicinity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/184,287, filed Jun. 25, 2015, which is hereby incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION 1) Field of the Invention

The field of the invention is immunology.

2) Description of Related Art

The skin can be a uniquely immunogenic target for immunization, as itcontains an extensive array of immunologically responsive cell typesthat contribute to both innate and adaptive immunity. Strategicengineering of the skin to create a pro-immunogenic microenvironmentcould lead to more effective preventive and therapeutic vaccines againstdreaded diseases such as cancer and AIDS.

The transcription factor x-box binding protein 1 (spliced) XBP1, whichis an endoplasmic reticulum (ER)-stress associated factor regulating ERstructure and function, can promote the production and secretion ofproteins, and regulate cell differentiation and survival in certaincells. However, the regulatory networks affected by XBP1 are not wellunderstood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A-D) contains graphs showing XBP1 enhances the production ofsecreted vaccine antigen and pro-inflammatory cytokines bykeratinocytes. OVA (FIG. 1A), IL-1α (FIG. 1B), IFN-β (FIG. 1C), CCL2(FIG. 1D).

FIG. 2(A-F) contains graphs showing that XBP1 promotes lymphocyte andCD11c⁺ cell infiltration into skin. FIG. 2A-B, untreated. FIG. 2C-D,Control vector+OVA. FIG. 2E-F, XBP1+OVA.

FIG. 3(A-F) contains graphs showing that XBP1 in vivo increases theexpression of pro-inflammatory cytokines and chemokines and co-deliveredvaccine antigen transgene. XBP1 (FIG. 3A), OVA (FIG. 3B), CCL2 (FIG.3C), TNF-α (FIG. 3D), IL-1α (FIG. 3E), IL-1β (FIG. 3F).

FIG. 4(A-E) contains graphs showing that XBP1 overexpression enablesinduction of durable systemic antigen-specific IFN-γ- and GranzymeB-expressing CD8⁺ T cell immunity. FIG. 4B showing untreated,restimulation with OVA-specific SIINFEKL. FIG. 4C showing controlvector+OVA, restimulation with OVA-specific SIINFEKL. FIG. 4D showingXBP1+OVA, restimulation with OVA-specific SIINFEKL, FIG. 4E showingXBP1+OVA, restimulation with β-gal-specific DAPIYTNV.

FIG. 5(A-F) contains graphs showing that XBP1 promotes the accumulationof memory [central (CD44⁺CD62L⁺) and effector (CD44⁺CD62L⁻)] CD8⁺ Tcells and skin-resident CD103⁺CD8⁺ memory T cells in skin at theimmunization site. FIG. 5A-B, untreated. FIG. 5C-D, Control vector+OVA.FIG. 5E-F, XBP1+OVA.

FIG. 6(A-O) contains graphs showing that overexpression of XBP1 in situ(FIG. 6A) triggers expression of known XBP1 responsive genes (GRP78,GFAT-1 and VEGFA; FIGS. 6B, 6C, 6D), and genes associated withkeratinocytes migration, proliferation and function (HIF-1α FIG. 6E),pro-inflammatory responses (IL-23α, S100A7, IL-1β, MyD88, OAS1, TNF-α;FIGS. 6F, 6G, 6I, 6K, 6N), the recruitment and activation of immunocytes(CCL19, CD86 and IL-15; FIGS. 6H, 6L, 6M), and co-delivered vaccineantigen transgene (OVA; FIG. 6O).

FIG. 7 contains a graph showing that transient overexpression of XBP1 inskin drives vaccine-induced durable protective immunity. Untreated(filled squares), control vector plus OVA (open circles), XBP1 plus OVA(open squares).

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compositions and methods for increasing an immuneresponse in a subject, immunizing a subject and/or treating a disease ina subject that relate to keratinocytes. Terms used throughout thisapplication are to be construed with ordinary and typical meaning tothose of ordinary skill in the art. However, Applicant desires that thefollowing terms be given the particular definition as defined below.

Definitions

As used in the specification and claims, the singular form “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The term “administering” refers to an administration that is oral,topical, intravenous, cutaneous, subcutaneous, transcutaneous,transdermal, intramuscular, intra-joint, parenteral, intra-arteriole,intradermal, intraventricular, intracranial, intraperitoneal,intralesional, intranasal, rectal, vaginal, by inhalation or via animplanted reservoir. The term “parenteral” includes subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional, and intracranialinjections or infusion techniques. In one embodiment, the administrationis cutaneous or transdermal. It should be understood that a cutaneousadministration does not require systemic delivery of the administeredcomposition.

The terms “about” and “approximately” are defined as being “close to” asunderstood by one of ordinary skill in the art. In one non-limitingembodiment, the terms are defined to be within 10%. In anothernon-limiting embodiment, the terms are defined to be within 5%. In stillanother non-limiting embodiment, the terms are defined to be within 1%.

The term “antigen” refers to any composition toward which an immuneresponse is generated. Antigens include, but are not limited to,polypeptides, oligopeptides, and polysaccharides. In one embodiment, theantigen is a polypeptide.

The terms “cell,” “cell line,” and “cell culture” include progeny. It isalso understood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenywithin a population, which population has the same overexpression ofXBP1 as screened for in the originally engineered cell population, areincluded.

A “composition” is intended to include a combination of active agent oragents (for example, an XBP1 pathway upregulating composition) andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

A “control” is an alternative subject or sample used in an experimentfor comparison purpose. A control can be “positive” or “negative.”

The term “disease” refers to an abnormal condition of a part, organ, orsystem of a subject resulting from various causes, such as infection,inflammation, environmental factors, or genetic defect, andcharacterized by an identifiable group of signs, symptoms, or both. Insome embodiments, the disease is a cancer.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

As used herein, “gene expression” and “protein expression” refer to theprocess by which polynucleotides are transcribed into mRNA and theprocess by which the transcribed mRNA is subsequently being translatedinto peptides, polypeptides, or proteins, respectively. If thepolynucleotide is derived from genomic DNA, expression may includesplicing of the mRNA in a eukaryotic cell. “Gene overexpression” refersto the overproduction of the mRNA transcribed from the gene, at a levelthat is about 2.5 times higher, about 5 times higher, or about 10 timeshigher than the expression level detected in a control sample. “Proteinoverexpression” includes the overproduction of the protein productencoded by a gene at a level that is about 2.5 times higher, about 5times higher, or about 10 times higher than the expression leveldetected in a control sample.

As used herein “surface expression” refers to the process by whichpolypeptides are translocated to the surface of a cell such that atleast a portion of the polypeptide is located at the exterior of thecell surface. “Surface overexpression” includes an increase in theamount of a particular polypeptide at the exterior surface of a cell, ata level that is about 2.5 times higher, about 5 times higher, or about10 times higher than the surface expression level detected in a controlsample.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular polypeptide or proteinafter being transcribed and translated. Any of the polynucleotidessequences described herein may be used to identify larger fragments orfull-length coding sequences of the gene with which they are associated.Methods of isolating larger fragment sequences are known to those ofskill in the art.

“Homologs” are defined herein as two polynucleotides or two polypeptidesthat have identity or homology. Homologs include allelic variants,orthologs, and paralogs having the same relevant function (e.g., abilityto upregulate the XBP1 pathway). In some embodiments, homologs haveabout 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92, 91% or 90% homology. Inother embodiments, homologs have about 80% or about 85% homology.

The term “identity” or “homology” shall be construed to mean thepercentage of nucleotide bases or amino acid residues in the candidatesequence that are identical with the bases or residues of acorresponding sequence to which it is compared, after aligning thesequences and introducing gaps, if necessary to achieve the maximumpercent identity for the entire sequence, and not considering anyconservative substitutions as part of the sequence identity. Neither N-nor C-terminal extensions nor insertions shall be construed as reducingidentity or homology. A polynucleotide or polynucleotide region (or apolypeptide or polypeptide region) that has a certain percentage (forexample, 80%, 85%, 90%, or 95%) of “sequence homology” to anothersequence means that, when aligned, that percentage of bases (or aminoacids) are the same in comparing the two sequences. This alignment andthe percent homology or sequence identity can be determined usingsoftware programs known in the art. In one embodiment, defaultparameters are used for alignment. In one embodiment a BLAST program isused with default parameters. In one embodiment, BLAST programs BLASTNand BLASTP are used with the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR.

The term “immunizing” refers to increasing an antigen-specific immuneresponse in a subject.

The term “keratinocyte” refers to an epidermal cell that expresses oneor more keratin polypeptides. The term “keratinocyte” includeskeratinocytes at each stage of differentiation, but does not includecorneocytes. In some embodiments, the keratinocyte expresses oroverexpresses a keratin-5 polypeptide and a keratin-14 polypeptide. Inother embodiments, the keratinocyte expresses or overexpresses akeratin-1 polypeptide and a keratin-10 polypeptide. In still otherembodiments, the keratinocyte expresses or overexpresses a keratin-1polypeptide and a keratin-16 polypeptide.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including human, domestic and farm animals, nonhuman primates,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.

As used herein, the terms “neoplastic cells,” “neoplasia,” “tumorcells,” “tumor,” “cancer,” and “cancer cells” (used interchangeably)refer to cells which exhibit relatively autonomous growth, so that theyexhibit an aberrant growth phenotype characterized by a significant lossof control of cell proliferation (i.e., de-regulated cell division).Tumor cells can be malignant or benign. A metastatic cell or tissuemeans that the cell can invade and destroy neighboring body structures.In some embodiments, the cancer is a skin cancer such as a melanoma, abasal cell carcinoma or a squamous cell carcinoma.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a pharmaceutically acceptable carrier, inert oractive, making the composition suitable for diagnostic or therapeuticuse in vivo or ex vivo.

The term “pharmaceutically acceptable carrier” means a carrier orexcipient that is useful in preparing a pharmaceutical composition thatis generally safe and non-toxic, and includes a carrier that isacceptable for veterinary and/or human pharmaceutical use. As usedherein, the term “pharmaceutically acceptable carrier” encompasses anyof the standard pharmaceutical carriers, such as a phosphate bufferedsaline solution, water, and emulsions, such as an oil/water or water/oilemulsion, and various types of wetting agents. As used herein, the term“carrier” encompasses any excipient, diluent, filler, salt, buffer,stabilizer, solubilizer, lipid, stabilizer, or other material well knownin the art for use in pharmaceutical formulations and as describedfurther below. The pharmaceutical compositions also can includepreservatives. A “pharmaceutically acceptable carrier” as used in thespecification and claims includes both one and more than one suchcarrier.

The terms “pharmaceutically effective amount,” “therapeuticallyeffective amount,” or “therapeutically effective dose” refer to theamount of a composition such as an XBP1 pathway upregulatingcomposition, and optionally, antigen polynucleotide, that will elicitthe biological or medical response of a tissue, system, animal, or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician. In some embodiments, a desired response is a treatmentof a disease such as a bacterial infection, a viral infection or acancer such as a skin cancer. In some instances, a desired biological ormedical response is achieved following administration of multipledosages of the composition to the subject over a period of days, weeks,or years. The terms “pharmaceutically effective amount,”“therapeutically effective amount,” or “therapeutically effective dose”include that amount of a composition such as a XBP1 pathway upregulatingcomposition, and optionally, antigen polynucleotide, that, whenadministered, is sufficient to prevent development of, or alleviate tosome extent, one or more of the symptoms of the disease being treated.The therapeutically effective amount will vary depending on thecomposition such as the a XBP1 pathway upregulating composition, andoptionally, antigen polynucleotide, the disease and its severity, theroute of administration, time of administration, rate of excretion, drugcombination, judgment of the treating physician, dosage form, and theage, weight, general health, sex and/or diet of the subject to betreated. In the context of the present method, a pharmaceutically ortherapeutically effective amount or dose of a XBP1 pathway upregulatingcomposition, and optionally, antigen polynucleotide, includes an amountthat is sufficient to prevent development of, suppress the growth of, orreduce the numbers of, one or more skin cancer lesions.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably, and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers.

A polynucleotide may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also refers to both double- and single-stranded molecules.Unless otherwise specified or required, any embodiment of this inventionthat is a polynucleotide encompasses both the double-stranded form andeach of two complementary single-stranded forms known or predicted tomake up the double-stranded form. As used herein, an “antigenpolynucleotide” is a polynucleotide that encodes a corresponding antigenpolypeptide. A polynucleotide is composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T);and uracil (U) for thymine (T) when the polynucleotide is RNA. Thus, theterm “polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

The term “polypeptide” is used in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orpeptidomimetics. The subunits may be linked by peptide bonds. In anotherembodiment, the subunit may be linked by other bonds, e.g. ester, ether,etc. As used herein the term “amino acid” refers to either naturaland/or unnatural or synthetic amino acids, including glycine and boththe D or L optical isomers, and amino acid analogs and peptidomimetics.A peptide of three or more amino acids is commonly called anoligopeptide if the peptide chain is short. If the peptide chain islong, the peptide is commonly called a polypeptide or a protein.

The terms “prevent,” “preventing,” “prevention,” and grammaticalvariations thereof as used herein, refer to a method of partially orcompletely delaying or precluding the onset or recurrence of a diseaseand/or one or more of its attendant symptoms or barring a subject fromacquiring or reacquiring a disease or reducing a subject's risk ofacquiring or reacquiring a disease or one or more of its attendantsymptoms.

The term “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. In someembodiments, the subject is a human.

“Suppress” tumor growth indicates a curtailment of growth state whencompared to growth without contact with a XBP1 pathway upregulatingcomposition and antigen polynucleotide described herein. Tumor cellgrowth can be assessed by any means known in the art, including, but notlimited to, measuring tumor size, determining whether tumor cells areproliferating using a ³H-thymidine incorporation assay, or countingtumor cells. “Suppressing” tumor cell growth means any or all of thefollowing states: slowing, delaying, and stopping tumor growth, as wellas tumor shrinkage.

The terms “treat,” “treating,” “treatment” and grammatical variationsthereof as used herein, include partially or completely delaying,alleviating, mitigating or reducing the intensity of one or moreattendant symptoms of a disease and/or alleviating, mitigating orimpeding one or more causes of a disease. Treatments according to theinvention may be applied preventively, prophylactically, pallatively orremedially. Prophylactic treatments are administered to a subject priorto onset (e.g., before obvious signs of disease), during early onset(e.g., upon initial signs and symptoms of disease), or after anestablished development of disease. Prophylactic administration canoccur for several days to years prior to the manifestation of symptomsof an infection. In some instances, the terms “treat,” “treating,”“treatment” and grammatical variations thereof, include partially orcompletely reducing the size of a solid tumor or cancer lesion orreducing the number of solid tumors or cancer lesions as compared withprior to treatment of the subject or as compared with the incidence ofsuch symptom in a general or study population.

The term “vector” means a DNA construct containing a DNA sequence whichis operably linked to a suitable control sequence capable of effectingthe expression of the DNA in a suitable host. Such control sequencesinclude a promoter to effect transcription, an optional operatorsequence to control such transcription, a sequence encoding suitablemRNA ribosome binding sites, and sequences which control the terminationof transcription and translation. The vector may be a plasmid, a phageparticle, or simply a potential genomic insert. Once transformed into asuitable host, the vector may replicate and function independently ofthe host genome, or may in some instances, integrate into the genomeitself. A plasmid is the most commonly used form of vector, however, theinvention is intended to include such other forms of vectors which serveequivalent function as and which are, or become, known in the art.

The term “XBP1 pathway upregulating composition” refers herein to anycomposition that when administered to a keratinocyte, increases oractivates a constituent in an XBP1 regulated/responsive pathway. Forexample, downstream pathways known to be effected by XBP1 include thoseof the ER stress Gene Network including, for example, Sec24c, Sec31a,Sec23b, Sec24d, Sec61a1, Copg1, Copb2, Gosr2, Golgb1, Golga3, Arfgap3,Rpn2, Spcs3, Fasn, Hspa13, Surf4, Jnk, Mfn2, Atf6, Dnajc3, Pdia6, Pdia5,Pdia4, Rpn1, Os9, Hyou1, Csdc47, Stt3a, and Nlrx1. Other downstreampathways known to be effected by XBP1 include: Protein transportpathways including, for example, those effecting GRP78; Cell metabolismpathways including, for example, those effecting GFAT-1; Pathwayseffecting blood vessel growth including, for example, those effectingVEGFA; Pathways effecting triglyceride biosynthesis including, forexample, those effecting Agpat6, Fasn, Scd2, or Lpar1; Pathwayseffecting keratinocytes migration, proliferation and function including,for example, those effecting HIF-1α and IL-1α; Pathways effecting innateimmune signaling including, for example, those effecting MyD88, OAS1,S100A7; Pathways effecting cytokine and chemokine expression including,for example, those effecting IL-1β, TNF-α, IFN-β, IL-6, IL-1α, IL-23α,IL-15, CCL2, CCL19, IL-12; Pathways effecting antigen presentationincluding, for example, those effecting Sec22B; CD40, CD70, CD86;Pathways effecting cell migration including, for example, thoseeffecting CCR7; Pathways effecting expression of transgenic antigensincluding, for example, OVA. In some embodiments, the term “XBP1 pathwayupregulating composition” refers herein to any composition that whenadministered to a keratinocyte results in increased innate immunestimulation, increased cytokine/chemokine expression, increased antigenprocessing and presentation function including those effecting theproduction of antigen, and/or increased expression or production of oneor more of IL-1α, IL-1β, IFN-β, TNF-α, IL-6, IL-12, IL-15, IL-23a, CCL2,CCL19, MyD88, OAS1, S100A7, Sec22B, CD40, CD70, CD86, CCR7 and HIF-1α inthe keratinocyte.

The term “XBP1” refers herein to an X-box binding protein 1 polypeptidealso known as Tax-Responsive Element-Binding Protein 5, TREB5, or XBP2,and in humans, is encoded by the XBP1 gene. The term “XBP1polynucleotide” refers to an XBP1 encoding polynucleotide and includesan XBP1 gene in its entirety or a fragment thereof. In some embodiments,the XBP1 polypeptide or polynucleotide is that identified in one or morepublicly available databases as follows: HGNC: 12801; Entrez Gene: 7494;Ensembl: ENSG00000100219; OMIM: 194355; and UniProtKB: P17861. In someembodiments, the XBP1 polynucleotide encodes an XBP1 polypeptidecomprising the sequence of SEQ ID NO:1, or a polypeptide sequence havingat or greater than about 80%, at or greater than about 85%, at orgreater than about 90%, at or greater than about 95%, or at or greaterthan about 98% homology with SEQ ID NO:1, or a polypeptide comprising aportion of SEQ ID NO:1. The XBP1 polypeptide of SEQ ID NO:1 mayrepresent an immature or pre-processed form of mature XBP1, andaccordingly, included herein are mature or processed portions of theXBP1 polypeptide in SEQ ID NO:1. In some embodiments, the XBP1polynucleotide comprises the sequence of SEQ ID NO:2 or a polynucleotidesequence having at or greater than about 80%, at or greater than about85%, at or greater than about 90%, at or greater than about 95%, or ator greater than about 98% homology with SEQ ID NO:2, or a polynucleotidecomprising a portion of SEQ ID NO:2.

DETAILED DESCRIPTION

Provided herein are compositions and methods for increasing an immuneresponse in a subject, immunizing a subject, and/or treating a diseasein a subject that relate to keratinocytes. It is a surprising finding ofthe present invention that modulation of an XBP1 pathway creates akeratinocyte that is itself an immune modulator, or adjuvant. In someembodiments, the concentration of antigen is increased in the vicinityof the keratinocyte, further increasing the immune response by effectorcells within that vicinity. Antigen concentration may be increased viaincreasing production of the antigen by the keratinocyte itself,increasing production of the antigen by another cell, or byadministering the antigen to the vicinity. In some embodiments, anantigen encoding polynucleotide is introduced into the keratinocyte andan XBP1 pathway upregulating composition is administered to thekeratinocyte to create a keratinocyte that is an antigen-specific immunemodulator. These modified keratinocytes produce extracellular antigen,and in some embodiments, pro-inflammatory mediators that facilitate anantigen-specific immune response by immune effector cells. The modifiedkeratinocytes described herein and the compositions contained thereinare useful for increasing immune responses, immunizing and treatingdiseases in subjects.

More specifically, the results provided in herein demonstrate thattransient overexpression of XBP1 locally in the skin microenvironment atthe time of antigen delivery induces potent local and systemic immuneresponses. Novel data is provided that demonstrates that inclusion ofplasmid DNA encoding XBP1 in skin results in significantly increaseddurable CD8⁺ T cell immune responses to a co-delivered transgenicantigen, and, interestingly, accumulated systemic memory and residentCD8⁺ T cells in the skin at the immunization site. XBP1's expression inthe skin is associated with the induction of a pro-inflammatorycutaneous microenvironment, as evidenced by increased expression ofpro-inflammatory cytokines and chemokine, and infiltration oflymphocytes and antigen presenting cells. Further, in the in vitrosystems, it is shown herein that XBP1 has a decisive role in promotingkeratinocytes to increase the production of co-delivered secretedantigen and one or more of IL-1α, IL-1β, IFN-β, TNF-α, IL-6, IL-12,IL-15, IL-23a, CCL2, CCL19, MyD88, OAS1, S100A7, Sec22B, CD40, CD70,CD86, CCR7 and HIF-1α. The present disclosure therefore describes forthe first time that XBP1 may function as a master transcriptionalregulator of keratinocytes to enhance the production of secreted vaccineantigens and pro-inflammatory mediators, resulting in a pro-immunogenicskin microenvironment that enables the induction of robust, durable andeffective local and systemic antigen-specific immune responses againstcancer or infectious diseases.

Accordingly, provided herein are keratinocytes comprising an XBP1pathway upregulating composition. In some embodiments, the keratinocytesfurther comprise an antigen polynucleotide. Included herein arekeratinocytes at any stage of differentiation, other than corneocytes.In some embodiments, the keratinocyte expresses or overexpresses akeratin-5 polypeptide and a keratin-14 polypeptide. In otherembodiments, the keratinocyte expresses or overexpresses a keratin-1polypeptide and a keratin-10 polypeptide. In still other embodiments,the keratinocyte expresses or overexpresses a keratin-1 polypeptide anda keratin-16 polypeptide. The keratinocytes produce an increased amountof the antigen extracellularly as compared to a control. In someembodiments, the keratinocytes produce an increased amount of one ormore pro-inflammatory mediators extracellularly as compared to acontrol. In one embodiment, the pro-inflammatory mediators are selectedfrom the group of IL-1α, IL-1β, IFN-β, CCL2, IL-15, IL23a, CCL19, andHIF-1α.

In some embodiments, the XBP1 pathway upregulating composition is avector comprising an XBP1 DNA polynucleotide. Expression of the XBP1 DNApolynucleotide in the keratinocyte results in overexpression of XBP1polypeptide as compared to a control, and thereby upregulates the XBP1pathway. Accordingly, included herein are XBP1 DNA polynucleotides,vectors comprising a XBP1 DNA polynucleotide, and keratinocytescomprising such vectors. However, the XBP1 upregulating composition maybe any composition that when administered to a keratinocyte, increasesor activates a constituent in an XBP1 regulated/responsive pathway. Insome embodiments, the XBP1 upregulating composition increasescytokine/chemokine expression, and/or increases antigen processing andpresentation function including those effecting the production ofantigen, and/or increases expression or production of one or more ofand/or increased expression or production of one or more of IL-1α,IL-1β, IFN-β, TNF-α, IL-6, IL-12, IL-15, IL-23a, CCL2, CCL19, MyD88,OAS1, S100A7, Sec22B, CD40, CD70, CD86, CCR7 and HIF-1α in thekeratinocytes.

In those embodiments in which the XBP1 pathway upregulating compositionis a vector comprising an XBP1 DNA polynucleotide, the vector may alsocomprise an antigen DNA polynucleotide. Accordingly, provided herein arevectors comprising an XBP1 DNA polynucleotide and an antigen DNApolynucleotide, and keratinocytes comprising such vectors. The antigenDNA polynucleotide may be linked to the same or a different promoter asthe XBP1 DNA polynucleotide. In one embodiment, the XBP1 DNApolynucleotide and the antigen DNA polynucleotide are operably linked tothe same promoter within the same vector. In other embodiments, the XBP1DNA polynucleotide and the antigen DNA polynucleotide are operablylinked to different promoters. The one or more promoters include, butare not limited to, a cytomegalovirus (CMV) promoter, a K14 (keratin 14)promoter, and a CD11c promoter. However, it should be understood thatthe present invention is not limited to the use of a specific promoter.Any promoter that achieves expression of the XBP1 DNA polynucleotidewithin a keratinocyte and/or the antigen DNA polynucleotide within arelevant mammalian cell is within the scope of this invention. In otherembodiments, the XBP1 DNA polynucleotide and the antigen DNApolynucleotide are contained within different vectors.

In some embodiments, the XBP1 polynucleotide encodes an XBP1 polypeptidecomprising SEQ ID NO:1 or a homolog or fragment thereof. In someembodiments, the XBP1 polynucleotide comprises SEQ ID NO:2 or a homologor fragment thereof. The antigen polynucleotide of the present inventioncan be any one known to those of skill in the art. In some embodiments,the antigen polynucleotide encodes a viral antigen. In otherembodiments, the antigen polynucleotide encodes a cancer-relatedantigen. In still other embodiments, the antigen polynucleotide encodesa bacterial antigen.

In other embodiments, the XBP1 upregulating composition affects anotherconstituent in the XBP1 pathway such as downstream effectors of XBP1including those of the ER stress Gene Network including, for example,Sec24c, Sec31a, Sec23b, Sec24d, Sec61a1, Copg1, Copb2, Gosr2, Golgb1,Golga3, Arfgap3, Rpn2, Spcs3, Fasn, Hspa13, Surf4, Jnk, Mfn2, Atf6,Dnajc3, Pdia6, Pdia5, Pdia4, Rpn1, Os9, Hyou1, Csdc47, Stt3a, and Nlrx1.Other downstream pathways known to be effected by XBP1 include: Proteintransport pathways including, for example, those effecting GRP78; Cellmetabolism pathways including, for example, those effecting GFAT-1;Pathways effecting blood vessel growth including, for example, thoseeffecting VEGFA; Pathways effecting triglyceride biosynthesis including,for example, those effecting Agpat6, Fasn, Scd2, or Lpar1; Pathwayseffecting innate immune signaling including, for example, thoseeffecting MyD88, OAS1; Pathways effecting cytokine expression including,for example, those effecting IL-1β, TNF-α, IFN-β, IL-6, IL-1α, IL-15,CCL2, IL-12; Pathways effecting antigen presentation including, forexample, those effecting Sec22B; CD40, CD70, CD86; Pathways effectingcell migration including, for example, those effecting CCR7; Pathwayseffecting expression of transgenic antigens including, for example, OVA.

Also provided herein are methods of treating or increasing an immuneresponse in a subject by administering to a keratinocyte apharmaceutically effective amount of one or more compositions comprisingan XBP1 pathway upregulating composition, wherein the keratinocyte is inthe subject or administered to the subject. In these methods, the XBP1pathway upregulating composition, and ultimately the keratinocyte,functions as an adjuvant to increase an immune response in the subject.The XBP1 pathway upregulating composition and the keratinocyte used inthe methods may be any as described above and below. In someembodiments, the XBP1 pathway upregulating composition is administeredto keratinocytes within the skin of the subject via a cutaneous, ortransdermal administration. In other embodiments, the XBP1 pathwayupregulating composition is administered to keratinocytes ex vivo, andthe keratinocytes are then administered to the subject via a form ofadoptive cell therapy.

In some embodiments, the method further comprises administering apharmaceutically effective amount of an antigen to the subject. In theseembodiments, the immune response to the antigen is increased, andtherefore, these embodiments include methods of immunizing the subjectto the antigen. The antigen may be administered before, after or at thesame time as the XBP1 pathway upregulating composition or keratinocytecomprising the XBP1 pathway upregulating composition. As describedherein, the antigen may be, but is not limited to, a polypeptide, anoligonucleotide, and a polysaccharide. The antigen may be administeredvia any means as described herein, and in some embodiments, isadministered via a cutaneous, or transdermal administration. In someembodiments, the antigen is administered to a site near or within thevicinity of the keratinocyte.

In some embodiments, an antigen polynucleotide is administered to thekeratinocyte. Accordingly, provided herein are methods of immunizing ortreating a subject by administering to a keratinocyte a pharmaceuticallyeffective amount of one or more compositions comprising an antigenpolynucleotide and an XBP1 pathway upregulating composition, wherein thekeratinocyte is in the subject or administered to the subject. In someembodiments, the antigen polynucleotide and the XBP1 pathwayupregulating polynucleotide are administered to keratinocytes within theskin of the subject via a cutaneous, or transdermal administration. Inother embodiments, the XBP1 pathway upregulating composition and theantigen polynucleotide are administered to keratinocytes ex vivo, andthe keratinocytes are then administered to the subject via a form ofadoptive cell therapy.

Administration of the antigen polynucleotide and an XBP1 pathwayupregulating composition to the subject results in an immune response inthe subject that is specific for the antigen. The antigen specificimmune response is mediated, at least in part, by CD8⁺ T cells. It isbelieved that, upon production of the antigen by the keratinocyte,antigen presenting cells present the antigen to CD8⁺ T cells, thusinitiating a CD8⁺ T cell antigen specific immune response. Anyconcomitant production of pro-inflammatory mediators by the keratinocytefacilitate this process. In some embodiments, antigen-specific IFN-γ-and Granzyme B-expressing CD8⁺ T cells are increased locally orsystemically in response to the administration. In other or furtherembodiments, CD103⁺CD8⁺ memory T cells are increased locally orsystemically in response to the administration. Since the methodsdescribed herein result in both effector and memory T cell responses,the methods of the present invention include use of the compositionsdescribed herein for both treatment and immunization.

Treatments according to the invention may be applied preventively,prophylactically, pallatively or remedially. Prophylactic treatments areadministered to a subject prior to onset (e.g., before obvious signs ofdisease or cancer), during early onset (e.g., upon initial signs andsymptoms of disease or cancer), or after an established development of adisease or cancer. Prophylactic administration can occur for severaldays to years prior to the manifestation of symptoms of the disease orcancer. In some instances, the terms “treat,” “treating,” “treatment”and grammatical variations thereof, include partially or completelyreducing the size of a solid tumor or a cancerous lesion or reducing thenumber of solid tumors or cancerous lesions as compared with prior totreatment of the subject or as compared with the incidence of suchsymptom in a general or study population. Accordingly, the methods oftreatment may comprise adoptive cell therapies (ACT) or vaccinationtherapies.

Included herein is a medicament for increasing an immune response in asubject in need thereof, comprising a pharmaceutically effective amountof a composition comprising an XBP1 pathway upregulating composition.Further included is a medicament for treating a viral infection, abacterial infection or a cancer in a subject, comprising apharmaceutically effective amount of a composition comprising an XBP1pathway upregulating composition. Still further included herein is a useof an XBP1 pathway upregulating composition in the manufacture of amedicament for the treatment of a viral infection, a bacterial infectionor a cancer. In each of these embodiments, the XBP1 pathway upregulatingcomposition can be as described above and below. In some embodiments,the medicament further comprises an antigen. The antigen can be any asdescribed above and below.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theinvention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof, which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims. Allpatents, patent applications, and publications referenced herein areincorporated by reference in their entirety for all purposes.

Examples Example 1: XBP1 Enhances the Production of Secreted VaccineAntigen and Pro-Inflammatory Cytokines by Keratinocytes

Pam 212 (a mouse keratinocyte cell line) cells (5-6×10⁴) were culturedovernight in 1 ml DMEM supplemental with 10% FBS, glutamine (2 mM) and1× antibiotic antimycotic solution and then untreated or transientlyco-transfected by plasmid DNA encoding Ova and XBP1 (or control vectorpcDNA3.1⁺) using the TransIT®-Keratinocyte Transfection Reagent (Mirus)according to vendor's instruction. After 3 days, Ova in the culturesupernatants were measured by ELISA (USCN life science Inc., HölzelDiganostik, Germany). The results are shown in FIG. 1 a.

FIG. 1(B-D) further shows that XBP1 enhances the production ofpro-inflammatory cytokines and chemokines by keratinocytes. Experimentswere performed as described in relation to FIG. 1A. After 3 days oftransfection, IL-1α, IFN-β and CCL2 in the culture supernatants weremeasured by ELISA. Data from four independent experiments are shown inFIG. 1(B-D) and were statistically analyzed (Student's t test). *P<0.05; ** P<0.01. Notably, in all cases, co-transfection of thedominant-negative XBP1, dnXBP1, which specifically inhibits XBP1swithout affecting cell viability or growth abrogated these XBP1soverexpression effects.

Example 2: XBP1 Promotes Lymphocyte and CD11c⁺ Cell Infiltration intoSkin

C57BL/6 (B6) mice (6-8 weeks, female) were untreated or immunized withOVA plus control vector DNA or OVA plus XBP1 DNA. 3 days later, skin atthe immunization site was removed and cut into small pieces andsubsequently incubated with Collagenase D (1 mg/ml) and DNase (1 mg/ml)in IMDM for 1 hour in an incubator, and then mashed and passed through a70 μm cell strainer. Single-cell suspensions of skin tissues werepre-incubated on ice with Fc Block (BD Bioscience) for 15 minutes andthen stained with Fixable Viability Dye EFLUOR® 780, anti-mouse CD45-APCand CD11c-PE-Cy7 and analyzed by flow cytometry using a BD LSRII flowcytometer (BD Biosciences) and analyzed using FlowJo software (v9.2,Tree Star). One representative of three independent experiments showingCD45⁺ (FIG. 2A, C, E) or CD11c⁺ cells (FIG. 2 B, D, F) among total liveskin cells is presented in FIG. 2.

Example 3: XBP1 Overexpression Increases the Expression ofPro-Inflammatory Cytokines and Chemokines in the Skin

Immunized skin was obtained as described in Example 2. Total RNA waspurified from homogenized skin using TRI REAGENT® (Sigma). RNA wasquantified by Nanodrop (Thermo Scientific). The cDNA was synthesizedfrom mRNA using The QIAGEN One-Step RT-PCR Kit with gDNA wipeout.Subsequently, TAQMAN® Assay-based real-time PCR was performed in anApplied Biosystems StepOnePlus™ Instrument following standard protocolswith primers purchased from IDT DNA for each gene. Mouse β-actin was acontrol. Relative mRNA expression was determined and normalized based onthe 2^(−ΔΔt) method. Data represent three mice from each group and werestatistically analyzed. The results are shown in FIG. 3(A-F).

Example 4: XBP1 Overexpression Enables Induction of Durable SystemicAntigen-Specific IFN-γ- and Granzyme B-Expressing CD8⁺ T Cell Immunity

B6 mice were untreated or immunized once as described in Example 3. 6-7weeks later, single-cell suspensions (3×10⁶) of splenocytes andvaccine/skin dLN) were restimulated with OVA-specific MHC I peptides(SIINFEKL) (or irrelevant β-gal MHC I peptides: DAPIYTNV) (2 μg/ml) in 2ml RPMI 1640 10% FBS for 3-4 days. IFN-γ in the culture supernatants wasdetermined by ELISA. Granzyme B was measured by surface staining ofanti-mouse CD8-Alexa flour 700 and subsequently intracellular stainingof anti-mouse granzyme B-Alexa-647 and analyzed by flow cytometry usinga BD LSRII flow cytometer and analyzed using FlowJo software. Onerepresentative of three independent experiments showing Granzyme Bexpression in gated CD8⁺ T cells is presented in FIG. 4(A-E).

Example 5: XBP1 Promotes the Accumulation of Memory [Central(CD44⁺CD62L⁺) and Effector (CD44⁺CD62L⁻)] CD8⁺ T Cells and Skin-ResidentCD103⁺CD8⁺ Memory T Cells in Skin at the Immunization Site

Mice were immunized as described in Example 4. 6-7 weeks later,single-cell suspensions from skin at the immunization site were preparedas described in Example 2, and subsequently pre-incubated on ice with FcBlock for 15 minutes and then stained with Fixable Viability Dye EFLUOR®450 (dead cells were excluded from analysis), anti-mouse CD45-APC,CD8-PE-Cy7, CD44-FITC, CD62L-percep5.5, CD103-PE, and analyzed by flowcytometry using a BD LSRII flow cytometer and analyzed using FlowJosoftware. One representative of 3 experiments showing CD44⁺CD62L⁺ andCD44⁺CD62L⁻ in gated CD8⁺ T cells or CD103⁺CD8⁺ T cells in gated totallive skin cells is shown in FIG. 5.

Example 6: XBP1 Increases Pro-Inflammatory Cytokines and Chemokines inHuman Skin In Situ

Human skin epidermal/dermal explants were left untreated or immunizedwith human XBP1 or control vector and cultured. 72 hours later, skin atthe immunization site was used for RNA extraction. Relative expressionof various genes was determined by real-time qRT-PCR and normalizedbased on the 2^(−ΔΔCt) method. The human B2M housekeeping gene served asan internal control.

Data from three experiments using three different human skin explants ispresented individually in FIG. 6(A-O), which shows increased expressionof XBP1, GRP78, GFAT-1, VEGFA, HIF-1α, IL-1β, MyD88, IL-23α, OAS1,S100A7, TNF-α, CCL19, CD86, IL-15, and OVA.

Example 7: Transient Overexpression of XBP1s in Skin DrivesVaccine-Induced Durable Protective Immunity

B6 mice (4/group) were untreated or immunized once as in Example 4. 5months later, mice were i.d. challenged with exponentially growingB16-OVA (1×10⁵). Melanoma growth was monitored and data werestatistically analyzed (9). * P<0.05. The results are shown in FIG. 7.

SEQUENCES SEQ ID NO: 1    1mvvvaaapsa ataapkvlll sgqpasggra lplmvpgpra agseasgtpq arkrqrlthl   61speekalrrk lknrvaaqta rdrkkarmse leqqvvdlee enhklqlenq llrekthglv  121venqelitrl gmdtldpdev peveakgsgv rlvagsaesa agagpvvtsp ehlpmdsdtv  181assdsesdil lgildkldpv mffkcpspes asleelpevy pegpsslpas lslsvgtssa  241kleainelir fdhvytkplv leipsetesq tnvvvkieea plssseedhp efivsvkkep  301leddfipelg isnllssshc lrppscllda hsdcgyegsp spfsdmsspl gtdhswedtf  361anelfpqlis v SEQ ID NO: 2    1atggtggtgg tggcagcggc gccgagcgcg gccacggcgg cccccaaagt gctactctta   61tctggccagc ccgcctccgg cggccgggcg ctgccgctca tggtacccgg tccgcgggca  121gcagggtcgg aggcgagcgg gacaccgcag gctcgcaagc ggcagcggct cacgcacctg  181agcccggagg agaaagcgct gcggaggaaa ctgaaaaaca gagtagcagc gcagactgct  241cgagatagaa agaaagcccg gatgagcgag ctggagcagc aagtggtgga tttggaagaa  301gagaaccaca aactccagct agaaaatcag cttttacggg agaaaactca cggccttgtg  361gttgagaacc aggagttaag aacacgcttg ggaatggaca cgctggatcc tgacgaggtt  421ccagaggtgg aggccaaggg gagtggagta aggctggtgg ccgggtctgc tgagtccgca  481gcaggtgcag gcccagttgt cacctcccca gaacatcttc ccatggactc tgacactgtt  541gcctcttcag attctgagtc tgatatcctt ttgggcattc tggacaagtt ggaccctgtc  601atgtttttca aatgtccttc cccagagtct gctagtctgg aggaactccc agaggtctac  661ccagaaggac ctagttcctt accagcctcc ctttctctgt cagtggggac ctcatcagcc  721aagctggaag ccattaatga actcattcgt tttgaccatg tatacaccaa gcctctagtt  781ttagagatcc cctctgagac agagagtcaa actaacgtgg tagtgaaaat tgaggaagca  841cctctaagct cttcagaaga ggatcaccct gaattcattg tctcagtgaa gaaagagcct  901ttggaagatg acttcatccc agagctgggc atctcaaacc tgctttcatc cagccattgt  961ctgagaccac cttcttgcct gctggacgct cacagtgact gtggatatga gggctcccct 1021tctcccttca gtgacatgtc ttctccactt ggtacagacc actcctggga ggatactttt 1081gccaatgaac ttttccccca gctgattagt gtctaa

1. A method of increasing an immune response in a subject in needthereof, comprising administering to a keratinocyte a pharmaceuticallyeffective amount of a composition comprising an XBP1 pathwayupregulating composition, wherein the keratinocyte is in the subject oradministered to the subject.
 2. (canceled)
 3. The method of claim 1,wherein the subject has a skin infection or a skin cancer.
 4. The methodof claim 1, further comprising administering a pharmaceuticallyeffective amount of an antigen to the subject.
 5. The method of claim 4,wherein the antigen is a bacterial antigen.
 6. The method of claim 4,wherein the antigen is a viral antigen.
 7. The method of claim 4,wherein the antigen is a cancer-related antigen.
 8. The method of claim1, wherein the XBP1 pathway upregulating composition is a vectorcomprising an XBP1 polynucleotide.
 9. The method of claim 8, wherein theXBP1 polynucleotide encodes a polypeptide comprising SEQ ID NO:1 or ahomolog thereof.
 10. The method of claim 8, wherein the XBP1polynucleotide comprises SEQ ID NO:2.
 11. The method of claim 4, whereinthe antigen is a polypeptide.
 12. The method of claim 11, wherein theXBP1 pathway upregulating composition is a vector comprising an XBP1polynucleotide and the vector further comprises an antigenpolynucleotide.
 13. The method of claim 21, wherein the subject istreated prophylactically.
 14. The method of claim 1, whereinadministration is cutaneous or transdermal.
 15. A polynucleotidecomprising an XBP1 polynucleotide operably linked to a K14 promoter andan antigen polynucleotide operably linked to a promoter.
 16. A vectorcomprising the polynucleotide sequence of claim
 15. 17. A keratinocytecomprising the polynucleotide sequence of claim
 15. 18. The keratinocyteof claim 17, wherein the keratinocyte has increased expression of one ormore of IL-1α, IL-1β, IFN-β, CCL2, IL-15, IL23a, CCL19, HIF-1α GRP78,GFAT-1, TNF-α, VEGFA, HIF-1α, OAS1, S100A7, and CD86.
 19. (canceled) 20.(canceled)
 21. A method of treating a viral infection, a bacterialinfection or a cancer in a subject, comprising administering to akeratinocyte a pharmaceutically effective amount of a compositioncomprising an XBP1 pathway upregulating composition, wherein thekeratinocyte is in the subject or administered to the subject.
 22. Themethod of claim 21, wherein the XBP1 pathway upregulating composition isa vector comprising an XBP1 polynucleotide and an antigenpolynucleotide.
 23. The method of claim 22, wherein the XBP1polynucleotide encodes a polypeptide comprising SEQ ID NO:1 or a homologthereof.